Exploring the protective effects of subnormothermic normoxic mechanical perfusion of genetically modified porcine erythrocyte perfusate on ischemic and hypoxic brain injury in cynomolgus monkeys

Objective  To explore the protective effects of genetically modified porcine erythrocyte suspension as a subnormothermic normoxic mechanical perfusate on hypoxic-ischemic brain injury in cynomolgus monkeys caused by traumatic hemorrhage.

Methods  Cynomolgus monkeys were randomly divided into positive and negative control groups (a total of 3 monkeys, with 3 left cerebral hemispheres as the positive control group and 3 right cerebral hemispheres as the negative control group) and the subnormothermic perfusion group (n=3). The positive control group was directly sampled 1 hour after circulatory arrest, while the negative control group was placed at subnormothermic conditions for 6 hours after circulatory arrest. The subnormothermic perfusion group underwent 6 hours of subnormothermic normoxic mechanical perfusion of the bilateral common carotid arteries of the cynomolgus monkey hypoxic-ischemic brain injury model using genetically modified porcine erythrocyte suspension 1 hour after circulatory arrest. Before perfusion, cross-matching experiments were conducted between the six genetically modified pig and the cynomolgus monkeys. After the start of perfusion, the levels of routine blood indicators in the perfusate were detected at 0, 1, 2, 3, 4, 5 and 6 hours. Blood oxygen saturation was recorded, and the levels of Na⁺, K⁺, Ca²⁺, glucose and blood pH in the perfusate were measured, as well as the levels of IgG and IgM in the perfusate. After 6 hours of perfusion, the water content of the brain tissue was measured. Nissl staining was performed on the frontal cortex and hippocampal regions, and immunofluorescence staining was used to detect the expression of glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba1), and neuronal nuclear antigen (NEUN).

Results  The cross-matching results between the six genetically modified pigs and the cynomolgus monkeys were negative. The number of red blood cells in the perfusate decreased significantly at 3 hours of perfusion, and the hemoglobin level showed a downward trend at 1, 3, 5 and 6 hours. The number of white blood cells and platelets decreased at all time points. The blood oxygen saturation in the subnormothermic perfusion group remained stable at 95%–98%, and the levels of blood oxygen saturation, Na⁺, Ca²⁺, glucose and pH were stable, while the K⁺ level first increased and then decreased. There was no significant difference in the levels of IgG and IgM before and after perfusion. The water content of brain tissue at the end of perfusion in the subnormothermic perfusion group was significantly higher than that in the positive control group (P<0.001). Nissl staining results showed that compared with the positive control group, the pyramidal neurons in the prefrontal cortex of the subnormothermic perfusion group maintained better morphological integrity, with no significant increase in enlarged and deformed cells. In the hippocampal CA1 region, there was a slight increase in enlarged and deformed cells, and a few cells with undamaged structures showed reduced cell size. In the hippocampal dentate gyrus, fewer granule neurons had compromised structural integrity, with increased cell edema. NEUN immunofluorescence staining showed that compared with the positive control group, the pyramidal neurons in the prefrontal cortex and hippocampal CA1 region of the subnormothermic perfusion group had better morphological states, with clear axons. The granule cells in the hippocampal dentate gyrus were well preserved, but the nuclei were less well protected. GFAP immunofluorescence staining showed that compared with the positive control group, the subnormothermic perfusion group had sparser protrusions that were more tightly associated with neurons. Iba1 immunofluorescence staining showed that compared with the positive control group, the subnormothermic perfusion group had thicker and fewer protrusions.

Conclusions  Compared with the positive control group, subnormothermic normoxic mechanical perfusion with genetically modified porcine erythrocyte perfusate increased brain tissue edema in cynomolgus monkeys, but better preserved the morphological integrity of neurons and glial cells. The protective effects may be related to the continuous oxygen and energy supply, maintenance of ion homeostasis and perfusate pH, reduced rejection, and low metabolic state of the whole brain.